Recently there has been increasing interest in the determination of the quantity and type of fatty acids present in the oils and fats which are used in the food, and other industries. For example, recently government and health organizations have called for, or imposed regulations on the amount and type of fatty acids contained in food products. Also, food suppliers are increasingly being mandated to provide information on the quantity and types of fatty acids contained within their products. This is particularly true for materials termed as “trans fatty” acids, as discussed hereinbelow.
Fats and oils are made of a complex mixture of a chemically similar group of compounds known as fatty acids. However, the composition of the fats and oils present in a given material is largely dependent on the source of the material. For example, vegetable oils are composed of mainly palmitic, palmitoleic, stearic, oleic, linoleic and linolenic acids. On the other hand, commercially manufactured “shortening” materials may contain over 30 different fatty acids including numerous trans fatty acids.
There are several different types of fat materials. Some fats occur naturally, while others are only attainable by diet. Briefly, the types of fat are saturated fats, unsaturated fats, phospholipids and triglycerides. Saturated fats are commonly found in animal fat products such as butter, lard and animal meats. Unsaturated fats, are divided into two groups, mono or poly unsaturated fats. An example of a monounsaturated fat is triolein, or its associated fatty acid, Oleic acid, which is the main component of olive oil. Polyunsaturated fats are essential fatty acids and are only attainable through diet. Examples of polyunsaturated fats are linoleic acid, linolenic, arachidonic acids, eicosapentaenoic acid (EPA) and decosahexaenoic acid (DHA). These fatty acids may be found in soy bean oil, peanut oil, corn oil, and fish oil, or more generally, oils extracted from fish, to name a few.
Phospholipid fats, the most common of which is lecithin, are an important common component of all cell membranes.
Triglycerides, are composed of three fatty acids attached to glycerol molecule and are, for example, the storage form of fat that occurs when humans eat calories in excess of their energy needs.
The so-called “trans fatty acids” are carboxylic acids with a long hydrocarbon chain in which the isolated double bond occurs in the “trans” configuration. It should be noted that most of the unsaturated sites in natural fats and oils from plant or animal origins generally occur in the “cis” double bond configuration. A small amount of “trans” fat is found naturally in ruminant fat, but is most commonly introduced into food or other materials during partial hydrogenation of, for example, vegetable oils. Levels of trans fat of up to 50% have been reported in products produced from partially hydrogenated vegetable oil.
This is of concern since recent studies have questioned the long term health issues related to the consumption of trans fatty acids. For example, studies have now suggested a link between trans fatty acid consumption and coronary heart disease. As such, there is increasing interest in determining the level of trans fatty acids in a material, and, more generally, in determining the quantity and type of all oils and/or fatty acids present in materials, and in particular, the levels and types of these materials which is present in food. Further, recent evidence has also suggested that there is a beneficial effect provided by EPA or DHA with respect to coronary heart disease, and it would therefore be useful to determine the levels of these, or other, beneficial materials.
Further, Omega-3 longer-chain (LC) polyunsaturated fatty acids (PUFAs), which are available mainly from fish oil in the form of triglycerides, have shown to have positive effect in reducing coronary heart disease (CHD). Since the body is unable to synthesize some of these Omega-3 (LC) PUFAs and they are required for improved health, these fatty acids have been termed ‘essential’ fatty acids. Two main Omega-3 (LC) PUFAs contained in fish oil are Eicosapentaenoic acid (EPA) and Decosahexaenoic acid (DHA), described hereinabove, and currently they are analyzed by gas chromatographic methods. As the health benefits of these fatty acids have increased over the last decade so has the commercial production and testing requirements.
Currently, the level and type of oils and/or fatty acid, and the determination of trans fatty acid, is performed using capillary gas chromatography (GC) analysis, or by use of infrared (IR) spectroscopic techniques, as described in a monograph from the American Oil Chemists' Society (“Official Method for the Determination of Trans Fat”, Mossoba et al., AOCS Press, 2003), the contents of which is incorporated herein by reference. While these techniques provide the necessary information, they suffer from some inherent difficulties.
First, the GC technique requires that representative samples be collected, processed and prepared for analysis (sometimes using toxic materials) over several hours, and then analyzed using a GC procedure that can take more than 45 to 60 minutes, or longer, to complete. As such, the GC technique can require several hours of a trained GC operator's time in order to finally prepare, analyze and report the results from the samples. In a production situation, the time and cost of this technique can be prohibitive.
This technique is also described by Satchithanandam et al. in “Trans, Saturated, and Unsaturated Fat in Foods in the United States Prior to Mandatory Trans-Fat Labeling”, Lipids, Vol. 39, No. 1 (2004).
Other chromatography techniques including silver ion Thin Liquid Chromatography (TLC-GC), and High Performance Liquid Chromatography (HPLC) are also known, but these techniques suffer from the same problems as the above mentioned GC techniques.
With the IR technique, a sample of the material to be tested is exposed to an Infrared light source, and the transmission or reflectance of the Infrared light is measured so that the amount of absorption can be determined. In traditional mid-infrared spectroscopy, the sample is progressively exposed to IR wavelengths so that an absorption spectrum is produced which can be compared to known standards for both absorption wavelength, and for the percentage of absorption. As such, the type of fatty acid or oil can be determined, and the amount present can be calculated.
This mid-infrared spectroscopy technique has been used since the 1940's for determining the trans content of fats and oils. However, it is also time consuming to prepare the sample for testing, and is subject to errors introduced by absorption of other materials such as water or the like. As such, using mid-infrared dispersive spectrometers that use prisms or diffraction gratings to resolve the infrared light into its component wavelengths, does not typically have the required accuracy necessary for precise quantitative analysis.
The advent of Fourier transform infrared spectroscopy (FTIR) has, however, led to improvements in compensating for absorption by other materials, and greatly improves the sampling time, and accuracy. In an FTIR machine, a pulse of infrared radiation is emitted, and an interferometer allows the essentially simultaneous detection of all of the reflected or transmitted component wavelengths of the mid-infrared region (4000 to 600 cm−1). A Fourier transformation calculation is then performed on the interferometer output to determine a spectrum which is essentially identical to the spectrum obtained by variation of the frequency.
Near Infrared (NIR) spectroscopy is a similar technique to infrared spectroscopy, wherein radiation from only the near infrared region is used. However, the interest in NIR for the analysis of various chemicals and other materials stems from a number of factors. For example, absorptions in the near infrared region arise from vibrational transitions to the second or higher energy states. Because of the very low probability of such transitions, absorption intensities are typically several orders of magnitude below those of the corresponding fundamental vibrations in the infrared and/or ultraviolet (UV) regions. Consequently NIR has improved sensitivity in the analysis of species present at low concentrations over conventional IR techniques.
Also, near infrared spectroscopy has the advantage that aqueous solutions can be readily analyzed without significant interference from water absorption since water does not significantly absorb the NIR radiation. Further NIR allows for the use of quartz or glass materials to be used in the construction of the NIR apparatus or in the sampling equipment, which materials cannot be used in traditional IR devices.
Further, the intense absorption of near infrared radiation at only selected wavelengths by a species, allows the NIR radiation to penetrate a sample sufficiently in order to be useful in the analysis of thicker samples.
As with the FTIR technique, Fourier transformation of the NIR spectrum (FT-NIR) provides improved results, wherein the FT-NIR instrument again makes use of an interferometer to encode data from the whole spectral range simultaneously. The interferometer, and preferably a Michelson interferometer, is thus used to produce a signal of a lower frequency than the frequency emitted from the NIR source. The lower frequency contains the same information as the original radiation signal, but its output is supplied at a speed slow enough for detection by a detector. The resultant output of the interferometer is an interferogram of all wavelengths emitted by the source.
A computer then performs a Fourier Transform on the interferogram and generates a frequency domain trace specific to the tested material.
FT-NIR spectroscopy has certain advantages over the traditional IR or NIR spectroscopy, in which the response of a sample to light is measured by scanning sequentially over a range of wavelengths. Primarily, however, the FT-NIR technique is rapid, less energy limited than using FTIR machines, can use glass or quartz cells, and can use sensitive detectors contained in more convenient forms. For example, FT-NIR devices are available which use fibre optics to transmit the NIR radiation to hand-held devices which can be merely inserted into the material to be tested. Alternatively, low cost glass sampling equipment can be used when analyzing the selected material.
FT-NIR spectroscopy has been previously used to determine the amounts of cis, trans, the relative degree of unsaturation or iodine values (IV), and the weight average molecular weight of saponification number (SN) parameters of edible fats and oils. This is described by Li et al. in “Rapid determination of cis and trans content, iodine value, and Saponification Number of Edible Oils by Fourier Transform Near-Infrared Spectroscopy”, JAOCS, Vol. 76, No. 4 (1999). However, this technique relies on establishing a series of known materials by using an analysis based on an FTIR technique, and using this information to establish a calibration curve for use with the FT-NIR device. However, this technique relies on the accuracy of the underlying FTIR technique.
A similar technique is described by Li et al. in “Trans Determination of Edible Oils by Fourier Transform Near-Infrared Spectroscopy”, JAOCS, Vol 77, No. 10 (2000), in which FT-NIR is used to measure trans fat content based on a calibration curved generated by testing a series of known samples using a single bounce, horizontal attenuated total reflectance, mid Infrared based technique.
While these techniques have some advantages over the prior art methods, they both rely on establishing a calibration curve (or matrix) based on a mid-FTIR technique, with its inherent analysis difficulties and accuracy limitations. To overcome these difficulties, it would be advantageous to provide a method of quantitative measurement of the amounts and types and/or categories of the fatty acid content in a material containing fats and oils, using a method with improved accuracy while maintaining a technique having good flexibility of use.